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Method for culturing osteoclast with mesenchyma stem cell combined with cell factor

A technology of mesenchymal stem cells and osteoclasts, applied in animal cells, tissue culture, vertebrate cells, etc., can solve the problems of no osteoclast culture method found, and solve the problem of difficult mass production, high maturity, and cost low effect

Inactive Publication Date: 2008-05-07
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] After searching, so far, no culture method that can provide a large number and high purity of osteoclasts has been found

Method used

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  • Method for culturing osteoclast with mesenchyma stem cell combined with cell factor
  • Method for culturing osteoclast with mesenchyma stem cell combined with cell factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] (1) Isolation and culture of mouse bone parenchymal mesenchymal stem cells

[0047] According to the inventor's patent of the present invention, mesenchymal stem cells were isolated and cultured from mouse bone parenchyma (Patent No. ZL200510055261.4) and the method disclosed by Guo Z et al. (Stem Cells.2006 Apr; 24(4):992-1000) For the isolation and cultivation of mouse bone parenchymal stem cells, 2-week-old healthy C57BL / 6 female mice (provided by the Animal Center of Military Medical Sciences) were taken, killed by neck dislocation, soaked in 75% alcohol for 5 minutes, and kept under sterile conditions Separate the mouse femur and tibia, remove the clean surface tissue, draw the phosphate buffer saline (weight to volume ratio: sodium chloride 8g / L, hydrogen phosphate) that contains 10% volume fetal bovine serum (purchase from Hyclone Company) Disodium 2.9g / L, Potassium Chloride 0.2g / L, Potassium Dihydrogen Phosphate 0.24g / L, solvent is deionized water) Rinse the bon...

Embodiment 2

[0061] Example 2 Determination of tartrate-resistant acid phosphatase

[0062] On the 3rd day, the 6th day, the 9th day, the 12th day and the 15th day of culturing respectively, according to the operating suggestion provided by the tartrate-resistant acid phosphatase assay kit (Sigma-Aldrich product) instructions, the culture wells of each group were The cells inside were stained, and each group stained 3 wells each time. The deep wine red cells were judged as TRAP(+) cells, and the TRAP(+) cells with more than 3 nuclei were judged as mature osteoclasts. The morphology was observed and photographed under an optical microscope. , and the counts of TRAP (+) cells and mature osteoclasts were analyzed statistically.

[0063] Result (see Fig. 1) the osteoclast obtained by the method of the present invention is compared with the osteoclast obtained by the cytokine method, the former is extremely active in morphology, with up to dozens of nuclei, huge cell body, and deep staining of ...

Embodiment 3

[0069] Example 3 Osteoclast Ivory Tablet Absorption Test

[0070] Ivory slices with a diameter of about 15 mm and a thickness of 3 μm (provided by Professor Jin Weifang, School of Medicine, Fudan University) were soaked in 75% alcohol for two hours, and sterilized by ultraviolet radiation in a clean bench for 12 hours. According to the above culture groups, mononuclear cells were planted in group D It was pre-placed at the bottom of the 24-well plate, and placed at the bottom of the 24-well plate before mesenchymal stem cells were laid in group F, and then mononuclear cells or mesenchymal stem cells and mononuclear cells were planted in sequence. Take out the ivory sheet on the 15th day, observe the lacuna formation situation on the surface of the ivory sheet under an optical microscope with toluidine blue staining and take pictures.

[0071] Results The mature osteoclasts obtained by the method of the present invention formed bone resorption lacunae on the ivory sheet (see Fi...

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Abstract

The invention discloses a culturing method of osteoclasts utilizing mesenchymal stem cells and cytikines, mainly utilizing the mesenchymal stem cells from mouse bone substance and the mouse splenic mononuclear cells and then adding nuclear factor kB receptor activator ligand and macrophage-colony to stimulate the factors. The invention has the advantages: the invention has the advantages of simpleoperation, convenience and practicality, capability of getting osteoclasts at the earliest time, and saving the culture time; the invention gets a large quantity of osteoclasts, solves the problem that osteoclasts are difficult to prepare in large scale, and can meet the need of a general cell biology experiment; the osteoclasts gotten through the invention has high maturity and strong bone substance absorptive capability, which is favorable for the function study; the cell factors needed by the invention is few, which saves expenditure.

Description

technical field [0001] The invention belongs to a method for culturing osteoclasts, in particular to a method for cultivating osteoclasts by using mesenchymal stem cells combined with cytokines. Background technique [0002] Osteoclasts (OC) are the main cells that absorb bone tissue in humans and animals. Under physiological conditions, osteoclasts and osteoblasts cooperate with each other to participate in bone reconstruction activities throughout the whole life process and maintain the integrity of the skeletal system. Dynamic balance; in pathological conditions such as tumors, inflammation, osteoporosis and autoimmune diseases, osteoclasts participate in the occurrence and progression of diseases, and affect the outcome and prognosis of diseases. [0003] Osteoclasts are differentiated and developed from hematopoietic stem cells, and can decompose bone tissue in vivo. Osteoclasts cultured in vitro can form resorptive lacunae on bone slices, ivory, or artificial bone tiss...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/077
Inventor 毛宁朱恒江小霞郭子宽
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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